The predicted Nef protein sequence of HIV-1, HIV-2 and SIV can be dissected into three structural regions: (a) leucine-repeat motif (dimerization domain); (b) autophosphorylation (kinase domain); and (c) acidic alpha-helical (transcriptional activation domain). Experiments were designed to define the functional domains of the Nef proteins of HIV- 1 and HIV-2. Genetic, biochemical and biological studies will be used to analyze the relationship between Nef structure and function. Preliminary results have shown that the HIV-2 (NIH-Z) Nef protein forms stable homo- oligomers in vitro and in vivo, including a phosphorylated homodimeric (50 kD) species. Site-specific mutagenesis studies of the conserved leucine repeat motif and cysteine residues within the N-terminal region of the Nef protein suggest that these residues have no direct influence on Nef oligomerization in vitro. In addition, glutathione S-transferase (GST) Nef fusions have been constructed for expression of the acidic transcriptional activation-like regions at the carboxy terminus of HIV-1 and HIV-2 (isolate SBL/ISY) Nef proteins, including an SH-2-type interaction sequence of the HIV-1 Nef protein, in E. coli. GST-Nef fusion proteins are used as Nef-affinity glutathione-Sepharose matrices to identify T cell factors that bind to, and are potential in vivo targets for HIV-1 and HIV-2 Nef proteins. Using GAL4-Nef fusions and transient transfection/CAT assays, identification will be made of regions of the HIV-1 Nef protein that may have transcriptional activation or repressive activity in COS-7 or HeLa cells. In addition, an endoplasmic reticulum (ER) retention-like signal sequence (I/VDDL) has been identified at the extreme carboxyl terminus of the HIV-1 Vpu protein. Site-specific mutagenesis was used to remove the terminal dipeptide sequence of the ER signal. Both wild-type and site-specific nef and vpu mutant genes were transfected into Jurkat E6-1 cells in the pCDNA-1 Neo vector for subsequent functional analysis.